Apomorphine sensitization: evoking conditions, context dependence, effect persistence and conditioned nature
S. Keller, J.D. Deliu s and M.J. Acerbo
Allgemeine Psycho logie, Universitat Konstanz, 78457 Konstanz, Germany
Correspondence to J.D. Delius, Allgemeine Psychologie, Universitat Konstanz, 78457 Konstanz, Germany.
E-mail: juan.delius@uni-konstanz.de
When repeatedly administered a dose of apomorphine (Apo), pigeons, much like rodents, show behavioural sensitization. In birds this sensitization expresses itself as an increasing pecking response to the drug and is found to be partially dependent on the environmental context in which Apo takes efl'ect. In the first experiment we examined what efl'ect difl'erent inter-Apo administration intervals have on the development of Apo sensitization and found that, with some smaller variations, intervals between 3 hours and 5 days all yielded comparable courses of sensitization. In the second experiment we examined how long pigeons had to be exposed to the same distinct cage to reveal a maximal context-dependent sensitization. Pigeons were therefore repeatedly injected with Apo and consistently placed in an experimental cage for difl'erent lengths of time (5 to 60 min; the overall drug efl'ect lasted for about 1 h) before being returned to their standard home cages. Subsequent tests in the experimental cage and a standard cage showed that 20-min Ilost-injection exposures were sufficient to yield a maximal response in the experimental cage. After training with 20-and 60-min exposures, the pigeons pecked about three times more in the experimental cage than in the standard cage. This confirmed the marked context dependency of the sensitization effect. In the third experiment, groups of pigeons were injected repeatedly with Apo and directly afterwards placed either consistently into the same experimental cage or into different experimental cages. The same-cage group evidenced a significantly much stronger sensitization than the difl'erent-cage group. A cage-habituation group served as a control for the possibility that the weaker sensitization of the different-cage group might be due to a cage novelty efl'ect. This cage-habituation group was run under the same conditions as the difl'erent-cage group but with additional exposures to the crucial cage while injected with saline. This extra treatment did not augment the pecking response to Apo in that cage. In the fourth experiment we examined how long the sensitization to Apo lasts and found that, even after 2 years of drug abstinence, it only waned to 50% of the original asymptotic response. The overall results sUPllort the hypothesis that a very major part of the sensitization to Apo in pigeons is due to a conditioning to the environmental context and to the drug state itself.
Keywords: pigeon, apomorphine, dopamine, pecking, sensitization, conditioning, memory
INTRODUCTION
Psychostimulant substances, several of which are abused by humans, are generally known to yield a sensitization effect when they are administered repeatedly to animal subjects. The effect involves an increase of the behavioural response to successive administrations of a given drug dose up to a ceiling response. Because it seems likely that the sensitization phenomenon relates to the development of the drug addiction connected with psychostimulants such as cocaine and amphetamine, it has been the subject of much research. The details of the processes respon- sible for the sensitization effect are nevertheless still a matter of some controversy. This undoubtedly is due
in part to the fact that different drugs, different doses, different procedures, different responses and different species or strains yield sensitization results that often differ in one or more respects. But some of the controversy has also arisen because there are several possible mechanisms that could be involved. Sensiti- zation might, for example, be due to a pharmaco- physiological up-regulation of transmitter release or of transmitter reception. This process could be modulated by a non-associative learning process, leading to a familiarization with the environmental context in which the drug takes effect. But sensitiza- tion could also be due to an associative classical conditioning of the drug response to external environmental cues or, indeed, to interoceptive cues
189 First publ. in: Behavioural Pharmacology ; 13 (2002), 3. - S. 189-201
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elicited by the drugs themselves. The sensitization might, alternatively, be brought about by a drug- rewarded instrumental conditioning of the responses elicited by the very same drug (see, for example, Hinson and Poulos, 1981; Kalant, 1989; Willner et al., 1992; Stewart and Badiani, 1993; Anagnostaras and Robinson, 1996; Mattingly ef aI., 1997; Adams
ef al., 2000; Crom bag ef al., 2000).
Cocaine and amphetamine, the substances with which most of the above-listed studies were carried out, are indirect and unspecific agonists of the neurotransmitter dopamine. Some of the complexities of the sensitization data that have given rise to the varied interpretations mentioned above may perhaps be due to this particular circumstance (Bedingfield et aI., 1996; Laudrup and Wallace, 1999). A number of direct and specific agonists of dopamine, although not known to be addictive, also give rise to behavioural responses in rodents akin to those elicited by cocaine and amphetamine, including a sensitization upon repeated administrations. The best studied of these substances is apomorphine (Apo), a potent D1- and D2-type dopamine receptor agonist.
Administered in doses of the order of 0.5-5 mgjkg to mice and rats, it elicits a bout of stereotyped oral responses (sniffing, licking, gnawing) and a concomi- tant period of increased locomotor activity, that under certain circumstances also expresses itself as climbing or turning behaviour (Moller et aI., 1987a;
Mattingly and Gotsick, 1989; Mattingly et al., 1997; Tirelli and Heidbreder, 1999a; Battisti et aI., 2000).
Most, but not all, studies on sensitization to Apo in rodents have focused on the locomotor responses because they are simple to measure with actometers or rotameters. However, these responses are not particularly specific to Apo, or indeed to psychosti- mulants generally, there being many other treatments that can elicit an analogous motor hyperactivity. We have, therefore, chosen to investigate the sensitization phenomenon in pigeons which, much as chickens and other avians, respond to Apo injections with a prolonged bout of repetitive pecking and little or no augmented locomotor activity. This response of birds, first described by Amsler in 1923, is in fact one of the most impressive examples of a drug eliciting an overt behavioural response. Even though it has been investigated repeatedly since it was discovered (e.g. Dhawan et aI., 1961; Brunelli et aI., 1975; Lanerolle and M illam, 1980; Machlis, 1980), our laboratory appears to be the first to have noticed tbat the pecking response to Apo was subject to sensi tization (Basten-Krefft, 1977; Deli us, 1985). The pecking response, which can be quantified easily and reliably, and which typically consists of several thousand pecks, is quite specific to Apo. Administra-
tion of other psychostimulant substances or even, more generally, other dopamine agonists, elicits at most only a few hundred pecks (Cheng ef aI., 1975;
Goodman, 1981; Idemudia and McMillan, 1984;
unpublished experiments). Comparably in tense bouts of pecking are otherwise only known to occur when very hungry pigeons are given access to a heap of ground grain mixed with coarse sand, or when hungry pigeons have been specially conditioned to peck for occasional grain rewards (Horster ef al., 2002). It must be noted though that Apo-induced pecking, although motorically very similar to forage pecking and, in fact, facilitated by food deprivation (Siemann and Delius, 1992; Wynne and Delius, 1995), is usually not directed at grains, since the drug has a pronounced hunger-suppressing side-effect (Deviche, 1984). Instead, Apo-induced pecking is mostly directed at small contrasting inedible features of the surrounding environment or even of the bird's own body surface (Keller and Delius, 2001).
In previous publications we have reported that repeated daily injections of between 0.2 and 2mgjkg Apo doses lead to a progressive increase of the pecking response up to a dose-dependent asymptote;
that this sensitization to Apo is markedly context- dependent; and that, in spite of some peculiarities, it is likely to be due to classical conditioning processes (Linden blatt and Delius, 1987; Wynne and Delius, 1995; Godoy and Delius, 1999; Godoy et aI., 2000; Keller and Delius, 200 I). In the experimen ts reported here, we first explored the effect of different temporal intervals between Apo treatments, ranging between a few hours and several days, on the development of sensitization to the drug. There is evidence that, in rats, shorter intervals between Apo injections lead to the development of tolerance, and that only longer intervals lead to the development of sensitization (Castro e/ aI., 1985; Casas et aI., 1999). Then we examined to what extent the sensitization arising in a distinct environment depended on the duration of the Apo-contingent exposure to that particular environ- ment. The background is that an injection of the standard dose of Apo (0.5 mgjkg) begins to have an effect after a few minutes, and yields a maximum rate of pecking some 10 min later. The response then gradually decreases to a level close to zero within approximately the next hour (Basten-Krefft, 1977;
Wynne and Delius, 1995). Next, we assessed the degree of the context dependence of the sensitization, by comparing the effect arising with treatments in a constant environment and in varying environments.
Using rats and amphetamine, Anagnostaras and Robinson (1996) found that although a constant environmental condition yielded the strongest sensi- tization, a varying condition still yielded an appreci-
able sensitization. Finally, we examined the degree to which the sensitization effect is retained over longer periods of Apo abstinence. In rodents, Apo treat- ments have been shown to have effects lasting several weeks (Mattingly ef aL., 1989; Battisti et at., 1999) but there are no data about the durability of the Apo sensitization in birds.
METHODS
General
Adult pigeons (CoLumba Livia), bred from local homing stock and weighing between 400 and 600 g, were used. New drug-naive pigeons were used in each experiment except the last one. For these experi- ments, the birds were moved from an outside aviary to individual stainless-steel grid cages (40 x 45 x 35cm) located in a well-lit (14 hours daily) and ventilated animal room. In these home cages the pigeons had free access to water and food. The experimental cages, which are described later, were located in a separate, well-lit room equipped with a video camera and recorder. Here the pigeons had no access to water or food. The number of pecks that each pigeon issued during the critical 20-min sessions were later counted by analysing the corresponding videotapes, with the aid of slow-motion replay when necessary. The drug treatments always involved 0.5mg/kg injections of racemic Apo (Teclapharm) into the pectoral muscle; the control treatments always involved injections of an equivalent volume of saline. All procedures used were in accordance with the rules and regulations of German animal protection law.
Treatment intervals
Sixty-nine pigeons were allocated randomly to seven groups of nine or 10 birds each. Each of the pigeons was injected with Apo and placed immediately afterwards into an experimental cage. The experi- mental cages were standard pigeon cages which had their sides and rear walls lined with white cardboard speckled with green dots of 8 mm diameter, at a density of about 10 dots per 100 C1112
. After 20 min in the experimental cages the pigeons were returned to the home cages. Depending on the group that they belonged to, they were treated again in the same manner after an in terval of either 3, 12, 16, 24, 48, 72 or 120 h. This was repeated for a total of six sessions.
The pigeons were videotaped during each of these sessions. Fourteen randomly selected birds out of the short-interval 3- and 12-hour groups, and 14 ran- domly selected birds out of the long-interval 72- and
120-hour groups, were treated in an analogous manner for three more sessions after a 3-week break.
Exposure durations
Thirty-two pigeons were allocated randomly to four groups of eight birds each. During a training phase, each of the pigeons was injected with Apo and placed immediately afterwards into an experimental cage, as described above. The pigeons remained there for a period of either 5, 10, 20 or 60 min, depending on the group to which they belonged. Afterwards, but while the behaviour of most pigeons was still under the influence of Apo (as the effect of the Apo dose mostly lasted for slightly more than 1 h), they were returned to their home cages, where they remained until their next daily session. The training procedure was repeated on five consecutive days. Each bird was treated at about the same time of the day throughout the experiment (cf. Arvanitogiannis et aL., 2000).
After a break of 2 days, tests took place on two successive days. After being injected with Apo, each pigeon was placed for 60 min into the experimental cage on the first test day and into a standard cage the next day. This latter cage was identical to the pigeons' home cage except that it lacked the food and water troughs and was located in the experimental room.
The pigeons were videotaped during all the training periods and during both test sessions.
Single and multiple contexts
Four different new experimental cages were used in this experiment (see Figure 3, top). Cage A was of cylindrical shape, 40 cm in diameter, 38 cm deep and the floor was 20 cm wide. The walls and the floor were painted white and speckled with green dots, 8 mm in diameter, about 10 per 100cm2. The front was made of wire netting. Cage B was a rectangular prismatic plastic container (29 x 51 x 25 cm), with rounded corners and edges made of transparent, pale-yellow plastic. The floor was a wooden platform covered with newspaper. Cage C was a standard, near-cubic (40 x 45 x 3S cm) cage with the floor, walls and ceiling lined with turquoise-coloured cardboard; the front was made of a stainless-steel grid. Finally, cage D was of a triangular prismatic shape, tapering towards the back (55 x 44 x 44 x 36 cm). The wire mesh front grid was painted white, the walls, floor and ceiling were lined with chessboard-like cardboard with black and white 7 x 7 cm squares.
Thirty-six pigeons were assigned randomly to three groups of 12 pigeons each. Each group was sub- divided in four subgroups of three pigeons each. The pigeons of the same-cage group were injected with Apo on four successive days and placed consistently into the same experimental cage according to the sequences AAAA, BBBB, CCCC or DDDD, depend- ing on the subgroup to which they belonged. The pigeons of the different-cage group also received Apo
on four consecutive days, but were placed into a different cage each day; the sequences for the four subgroups being ABCD, BCDA, CDAB and DABC.
After being videotaped in the corresponding cages for 20 min, the birds were returned to their home cages.
Each of the birds was exposed to the corresponding cages at about the same time of the day throughout the treatment sequences. The pigeons from the cage- habituation group were treated the same as the birds of the different-cage group, except that they were additionally injected with saline on each of the first 3 days and placed into the cage in which they were to experience Apo on their final day. This extra-cage habituation procedure was run according to the schedules ODD, AAA, BBB or CCC, depending on the subgroup to which they belonged. One-half of the pigeons received the habituation treatment about 4 h before and the other half about 4 h after their Apo- related treatment. When exposed to their last cage under the intl uence of A po, that particular cage was as familiar to them as the respective cages were to the same-cage group.
After their four sensitization sessions, the pigeons of same-cage group were placed into their corre- sponding cages for three daily 20-min sessions after having been injected with saline and videotaped in the usual way. The recording of an analogous treatment given to the pigeons of the different-cage group was marred by a video-recorder failure. The cage-habi- tuation group pigeons were not saline-tested.
Sensitization persistence
Fifty-four previously Apo-sensitized pigeons were used in this experimen t. Of these, 47 pigeons had participated in the treatment-interval experiment described earlier. The seven additional pigeons had been trained exactly as those of the 24-hour interval group of that experiment while serving in an experiment reported elsewhere (Acerbo, 200 I). All the pigeons had thus been previously sensitized to asymptotic responding with five or six injections of Apo and 20-min exposures to the green dots/white background experimental cages. The pigeons had then been returned to an outside aviary. Four or S days before the retention test, they were again housed in individual home cages. They were divided into groups, balanced, as far as feasible, for the various treatment conditions that they had been exposed to during the sensitization training. The groups were tested for the amount of pecking induced by the renewed administration of O.S mg/kg after experi- mental breaks lasting an average of 2.2 months (between 2 and 3 months, 33 pigeons), precisely 6 1110nths (nine pigeons), precisely 12 months (five pigeons) and an average of 23.3 months (between j 7
and 32 months, seven pigeons). The pigeons were placed for 20 min into the experimental cage and videotaped. The 23.3-111onth group birds were in fact first tested after having been injected with saline for one session and were only then subjected to a complete Apo-resensitization treatment over seven daily sessions.
RESULTS Treatment intervals
Five pigeons belonging to various groups exhibited extremely weak pecking reactions throughout. As subsequently discovered, they were all descended from a breeding lineage that consistently produced offspring nearly unresponsive to Apo (Keller, 2001; cf. Overstreet and Pucilowski, 2000 for data on rats).
These pigeons were excluded from the evaluation, so that the groups finally included between 8 and 10 pigeons each. The mean pecking responses
±
standard errors (SE) per group per session were computed. All groups showed the characteristic sensi tization curve.The curves of three representative treatment groups are shown in Figure I, upper panel. The differences between the groups were assessed using the mean pecking shown by each pigeon during the fifth and sixth sessions; that is, when the pigeons were exhibiting a near-asymptotic response. Averages ± SE of these mean scores were calculated for each treatment group and are shown in Figure I, lower panel. It can be seen that the shortest inter-session intervals of 3 and 12 h yielded asymptotic pecking scores that were about 1000 pecks higher than those corresponding to the longer inter-session intervals, ranging from 16 to 120 h, whereas the results of the 72-hour-interval group suggest a minor intermediate peak. Mann-Whitney tests showed that, except for the 72-hour group results, the differences between the short- and long-interval groups were all significant at the P < O.OS level.
The stronger response of the birds treated with shorter intervals (14 pigeons: 3- and 12-hour groups pooled), compared with those treated with longer intervals (14 pigeons: 72- and 120-hour groups pooled), present at the end of the original sensitiza- tion (3993± 144 and 3474± 167 pecks; Mann-Whit- ney, P<O.OS) was no longer present after a 3-week break (3781 ± 261 and 3981 ± 120 pecks; NS). How- ever, after a resensitization treatment over three sessions, the original difference re-emerged partially, but without quite reaching significance.
Exposure durations
Inspection of the videotapes recorded during the training periods, while the pigeons were in the
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SE near-asymptotic responding as a function of all the treatment intervals used (lower panel). The experimental cage is shown schematically.experimental cages, showed that the pigeons re- sponded to the Apo administrations with pecking responses evincing much the same time course as described before. It was also obvious that they showed increases in responding with the repeated Apo administrations; that is, that they developed a progressive sensitization to the drug. Direct observa- tions confirmed that the pigeons also continued to peck in their home cages for about just over 1 hour after each Apo injection. The cross-welds of the steel- grid home cages served as pecking targets (cf. Wynne and Delius, 1995). One pigeon belonging to the 60- min exposure group showed an extremely weak pecking reaction throughout. It also descended from the previously mentioned Apo-unresponsive lineage.
This pigeon's data were excluded from the evaluation we now describe.
The numbers of pecks that each pigeon emitted during the first 20 min of each of the test sessions were counted by analysing the videotapes. Figure 2
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shows the mean pecking scores
±
SE calculated from these data, and corresponding to the four groups during the test sessions in the experimental and the standard cages. The mean pecking scores obtained in either cage ranged from about 1000 to 3000 pecks per 20 min, indicating the incidence of sensitizations that ranged from weak to strong when compared with those illustrated in Figure I. The group with as-min training exposure to the experimental cage showed nearly identical, though relatively weak, responses in both cages. With the lO-min and 20-min training exposures to the experimental cage, increasing divergences between the mean pecking response shown in the two cages become apparent. Both the increase in experimental cage test pecking and the decrease in standard cage test pecking from the 5- to the 20-min training conditions were significant (Mann-Whitney tests, P < 0.05). The further in- creased experimental cage exposure of 60 min during training did not yield any further differentiation of test responding in the experimental and the standard cage. The differences between experimental and standard cage pecking following the 20- and 60-min training were, however, significant in each case (Wilcoxon tests, P < 0.05). Notice that the total mean pecking scores in the experimental and standard cageadded up to an almost constant 4000-odd pecks per 40-m i n overa II testi ng time, regard less of the d i fferen t training exposure conditions.
Contrary to what could perhaps be expected (cf.
Ohyama et al., 2001), mean peck counts, extending to the second and third 20-min periods of the 60-min test sessions belonging to the 5-min group and the 60- min group pigeons, yielded no significant evidence that the temporally patterned regimes of training had led to temporally modulated patterns of sensitized responding.
Single and multiple contexts
The data of one pigeon of the same-cage group had to be excluded because it was Apo-unresponsive, for the reasons descri bed earlier. A pigeon from the cage- habituation group also had to be excluded because it became ill during the experiment.
Figure 3 summarizes the results of the experiment.
The same-cage group showed a very marked sensiti- zation. The different-cage and the cage-habituation groups evinced considerably weaker sensitizations.
Comparisons of the pecking scores shown during the first and the fourth session showed the large increment of the same-cage group to be highly significant at the P<0.005 level (Wilcoxon tests), while the small increments of the other two groups were significant at the lower P < 0.05 level (Wilcoxon tests). A between-group comparison of these incre- ments showed that there was no significant difference between the different-cage group and the cage- habituation group (mean increment scores 651 ±278 and 703 ± 211 pecks, respectively), but that there were highly significant differences between both of these groups and the same-cage group (mean increment score 2065 ± 236 pecks; Mann-Whitney tests, P<0.005). Note that the training involving only four sessions, rather than the more usual five or six sessions, did not allow for fully asymptotic sensitiza- tion levels. The relatively large SEs associated with these scores are no doubt due to the fact that the different cages modulated the amount of pecking that Apo elicited in them. It is well established that the amount of pecking induced by Apo in pigeons, and also chickens, is influenced by different visual characteristics of the surrounding environments (Brunelli et at., 1975; Lanerolle and Millam, 1980;
Keller and Deli us, 2001). Nevertheless, even in the plainer cages, Band C, the pigeons showed a high incidence of pecking. The fact that the pecking scores of the first sessions of all groups were even somewhat higher than usual (see Figure I) indicates that the cages employed were suitably response supportive. The responses of the various subgroups dealing with different cages, or cage sequences, did
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not differ from each other sufficiently to warrant separate analyses.
When tested after having been injected with saline, the pigeons of the same-cage group responded with a mean rate of 54 ± 10 pecks per session. This is an obviously much weaker response than tbat shown in the same context by the same pigeons after Apo administration, but it conforms with the after-saline response obtained in other Apo-sensitization experi- ments (47±9 pecks). It is significantly higher than the after-saline response shown by unsensitized, saline- only trained control pigeons (10
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FIGURE 4. Mean percent retention ± SE ofthe sensitized pecking response to Apo after experimental breaks of increasing length. Four different groups of pigeons were Apo-treated to asymptote (100%) and were tested for retention after various experimental breaks.
Sensitization persistence
The pecking scores of each pigeon during the only (or first) retention test session under the influence of Apo were expressed as a percentage of the average number of pecks produced by the same pigeon during the last two sessions of training. This normalization served to neutralize the variations in asymptote due to the varying training conditions to which the pigeons had been subjected (see Figure 1). Figure 4 shows the means of these percentages
±
SE, plotted as a func- tion of the retention interval. It is noteworthy that the mean test scores drop gradually with the lengthening of the retention interval, but that even after almost 2 years, the pigeons still yielded a pecking response that amounted to 53±
7% of the original asymptotic sensitization. By the fifth day of being resensitized, these latter birds regained their original average asymptotic response scores. Their mean pecking response under the influence of saline (29±
21 pecks per 20 min) was also about midway between that obtained immediately after an Apo sensitization (47 ±9 pecks) and that pertaining to a control saline-only trained group (10±7; these latter being the same two scores mentioned above). A separate analysis did not reveal any significant differences in response retention that could be attributed confi- dently to the various treatment intervals used during the original sensitization training (see above, Treat- ment intervals).DISCUSSION
In a series of previous studies we have established that the sensitization to Apo, with respect to the
pecking response in pigeons, is importantly context- dependent. On the basis of this and additional evidence, we have argued that the sensitization most likely arises through a classical, Pavlovian condition- ing process (Linden blatt and Delius, 1987; Burg ef al.,
1989; Wynne and Delius, 1995; Godoy and Deli us, 1999; Keller and Delius, 200 I). This conditioning account assumes that the Apo administration in pigeons yields a protracted unconditioned stimulus (US) state that elicits a prolonged unconditioned repetitive pecking response (UR). The environmental context in which successive administrations of Apo have their effect functions as a conditioned stimulus (CS) that comes to elicit an increasing conditioned pecking response (CR), which adds progressively to the UR triggered by the drug. This brings about an increased response to the same Apo dose and also brings about the context dependency of the response increment, which are characteristic of the pigeons' sensitization to Apo. Furthermore, the account assumes that stimulus generalization is likely to be at least partly responsible for the sensitization components that prima facie appear to be context independent. These are the portions of the sensitiza- tion increments that become manifest in environ- ments that are apparently different from, but which on closer examination nevertheless share similarities with, that in which the pigeons were explicitly sensitized. The concept of stimulus generalization refers to the circumstance that it is a common finding in conditioning experiments that, on suitable testing, part of the CR transfers to stimuli that are physically or perceptually partly similar to the original training CS (Domjan, 1993). This account also assumes that injected Apo, besides having an undisputed US effect, also has an interoceptive CS effect, and that the overall context CS to which the pecking CR develops incorporates this Apo-elicited CS compo- nent as an inevitable part element. This means that the exteroceptive context stimuli are only fully effective as a CS triggering a pecking CR when they are combined with the interoceptive state induced by the administration by Apo. Exposures to the exteroceptive environment after control saline administration elicit a weaker, partial pecking CR (Wynne and Delius, 1995; Godoy and Delius, 1999;
Keller and Delius, 2001). In the same vein, the circumstance that a CS-no-US condition necessarily lacks the Apo-mediated interoceptive CS component also explains why no latent inhibition or response extinction effects can be demonstrated in connection with Apo sensitizations in pigeons (Wynne and Delius, 1995; Godoy and Delius, 1999). In more usual conditioning experiments, such repeated CS- no-US pairings always lead to a prospective or
retrospective weakening of the CR (Domjan, 1993).
In what follows we discuss the result of the present experiments in the light of the above conditioning account of Apo sensitization.
Treatment intervals
Successive repeated administrations of the same Apo dose, with inter-treatment intervals ranging from 3 hours to 5 days, all yielded pronounced sensitiza- tions. Contrary to what has been found in rats (Castro et al., 1985; Casas et aI., 1999; see also Mattingly et al., 1989), we obtained no evidence that shorter inter-injection intervals lead to tolerance instead of sensitization. Such an effect would have needed some ad-hoc accounting within our condition- ing hypothesis. However, an as yet unpublished experiment in which pigeons received Apo with intra-peritoneal osmotic pumps at a rate of approxi- mately 0.5 mg/kg Apo per day for 7 days revealed a markedly weaker sensitization when the birds were subsequently tested with intramuscular injections of 0.5 mg/kg Apo. This might indicate the development of some tolerance (cf. Nelson and Ellison, 1978; Post, 1980: rodents, amphetamine). Note, however, that when definite tolerance occurs in the context of other drugs, it has also been found to be modulated by context conditioning (Kim et al., 1999).
The shorter treatment intervals of 3 and 12 h actually produced a somewhat higher sensitization asymptote than the longer treatment intervals of 16, 24, 72 and 120 h in our experiments. Notice that because the three latter intervals are all multiples of 24 h, the corresponding pigeons were always injected at about the same time of day. The shorter intervals imply that injections took place at different times of day. It has been shown that the effect of Apo administration in rats is modulated by a diurnal rhythm (Nagayama et aI., 1978; Nakano et al., 1980;
see also Gaytan et aI., 1999: amphetamine), but it is unlikely that the differences between the shorter and longer interval are due to this factor, because the 16- hour group, which yielded a lower asymptote, was also injected at differing times. Another possibility is that the stress arising with more frequent injections might have potentiated the Apo effects. Stress, although arguably stronger and more lasting than mere i.m. injections, has been shown to enhance Apo- induced responses in rats (Csernansky et aI., 1984;
Cabib et al., 1988). However, the results of a separate experiment, in which this stress hypothesis was specifically examined in pigeons by treating them with supernumerary saline injections, did not yield any significant support (Keller, 200 I).
In rats, it has been found that the peak Apo plasma
concentration occurring some 5 min after an Apo i.p.
I11Jection is reduced to half within about 20 min, and that Apo bound to striatal neural tissue has a similar half-life (Martres et aI., 1977; Smith et al., 1979). Assuming that comparable Apo clearances apply in i.m. Apo-injected pigeons, then the stronger sensitization seen with the shorter treatment inter- vals can hardly be due to a bodily accumulation of Apo. Nevertheless, on being re-treated after a 3-week break, the pigeons treated with the shorter 3- and 12-hour intervals no longer revealed a higher mean rate of responding, compared with the pigeons treated with the longer 72- and 120-hour intervals. Thus, the difference between shorter- and longer-interval treatments seemed to arise because at first an Apo injection somehow leaves a rapidly decaying trace, that nevertheless lasts for somewhat longer than 12 h, which then turns into a weaker, more slowly decaying trace lasting at least 120 h.
The results show that distributed trials (long intervals) are as conducive to learning as the same number of massed trials (short intervals). While in humans the former training regime tends to generally lead to more conditioning than the latter, in animals this phenomenon is far less clear-cut (Domjan, 1993). The little evidence that is available concerning pigeons suggests that the two conditioning regimes make little or no difference to their learning performance (Xia et al., 1995).
Exposure duration
As the pigeons' exposure to the experimental cage under the influence of Apo increased from 5 min to 20 min, the pecking exhibited during the subsequent tests in this cage increased from about 2000 pecks to about 3000 pecks per 20 min. Comparisons with the sensitizations shown in Figure I indicate that the present sensitization levels ranged from medium to high. The results establish that experimental cage exposures of 20 min suffice for producing a maximal sensitization, since longer, 60-min training exposures did not yield stronger test responses. Training sessions lasting 20 min, but not less, are thus sufficient to assess the full extent of the sensitization to Apo, even though the standard 0.5 mg/kg Apo dose induces a pecking response that actually goes on for about 60 min or slightly longer.
At the same time, as the pigeons' exposure to their home cages under Apo during the training phase increased inversely from a few minutes to about an hour, the test pecking in the standard cage rose from about 1000 pecks to about 2000 pecks per 20 min.
The generally weaker sensitization revealed in the standard cage was undoubtedly mainly due to the fact that the exposure to the home cages under Apo
mai nly took place when the effect of the drug dose injected was already waning (equivalent to a reduced UR). However, it is also a fact that the pigeons experienced the home cages during extended periods (for about 23 h between sensitization sessions) while they were not under the influence of Apo. Such an extensive exposure to a CS-no-US condition could potentially have led to an inhibition and/or extinction effect that could have weakened the pecking CR developing in response to the home cage CS (Domjan, 1993). However, as explained at the beginning of this Discussion, inhibition and extinc- tion treatments have previously been found to have negligible effects on the Apo sensitization of pigeons.
It is also possible that a blocking of conditioning due to a ceiling effect might have intervened (Domjan, 1993' but see Ahmed ef al., 1998), signifying that within a given training session the initial experimental cage would capture most of the conditioning feasible, leaving little scope for a subsequent conditioning to the home cage. On the other hand, a generalization between the somewhat similar cages (identical form, floor and fronts) could have led to a pecking CR component that was expressed in both the experi- mental and standard cages used during the tests (cf.
Godoy and Delius, 1999).
While, with training exposures to the experimental cage increasing up to 20 min, an augmenting propor- tion of the sensitized response became tied to that particular cage, this trend did not continue when the relevant exposures were lengthened to 60 min. The above-mentioned ceiling/blocking mechanism could have intervened to truncate the pecking associated with the latter treatment. 1t seems possible that the conditioning achievable within a session was already exhausted after a 20-min exposure to the experi- mental cages. The fact that the mean test responses in both cages added up to a nearly constant overall response of 4000 pecks per 40 min, irrespective of the training conditions, suggests that there was indeed some overall ceiling to the conditioning. This max- imum responding cannot be ascribed to the highest frequency that pigeons can physically peck. Such a limitation can only be expected to playa role with response rates above some 3600 pecks per 20 min (Horster ef al., 2002).
Regardless of these details, the overall results confirm the context dependency of the Apo sensitiza- tion, inasmuch as it was importantly determined by the exposures to the two types of cages, even though all pigeons received the same regime of Apo injec- tions. If the sensitization induced had been context- independent, the pecking responses during tests should not have been affected by variations in cage exposure during training.
Single and multiple contexts
The results most directly relevant to the conditioning account are those of the single and mUltiple contexts experiment. The single, constant-cage CS treatment yielded sensitization increments roughly three times larger than the different, multiple-cages treatment.
The constant cages can be assumed to have func- tioned as consistent CSs, allowing the systematic build-up of increasing pecking CRs, while the different cages can be expected to have acted as diverse CSs that prevent the build-up of any sizeable CRs. The advantage of the same-cage treatment cannot be explained by the circumstance that the pigeons of this group had become more ha bituated to the particular cage to which they had been exposed.
A control group of pigeons also exposed to different cages under Apo, but separately habituated under saline to the critical final cage, did not exhibit any marked sensitization. Note that using rats and amphetamine, Anagnostaras and Robinson (1996) found that although a constant environmental con- dition yielded tbe strongest sensitization, a Illultiple environmental condition still yielded a sizeable sensitization. However, their multiple environment training involved a, perhaps critical, double exposure to the same rotameter context.
Our present results corroborate earlier findings that Apo-sensitized pecking was only fully evident in the same cage in which the sensitization had earlier taken place, but not in an alternative, quite different cage. The results also confirm that mere cage context familiarity plays at most a minor role in the sensitization to Apo in pigeons (Godoy and Delius, 1999). The results are also in line with the finding that, given a choice, pigeons will almost exclusively peck at stimuli belonging to the context in which they were sensi tized, and do not peck at stim uli belonging to an alternative control context in which they had been treated with saline (Keller and Delius, 2001).
Contrary to what Burg ef al. (1989) had surmised on the basis of a less elaborate experiment the different- cage-treated pigeons did not develop tolerance (see also under Treatment intervals).
The lesser, but still significant, response increments obtained in the different-cage and the cage-habitua- tion groups show that there is nevertheless a smaller component of sensitization that is apparently con- text-independent. We cannot exclude the possibility that this weaker sensitization component might be due to a physiopharmacological up-regulation of the efficacy of the drug. However, it must be kept in mind that, although the cages used with the two groups were different from Apo session to Apo session, the complete context linked with the successive Apo treatments was not wholly different. The handling
and the injection procedures, and the furnishings of the experimental room (video camera, lights, win- dows, tables, etc.), remained the same across the different-cage treatments. Also, not to be forgotten, the behavioural state induced by the drug was likely to be the same. These commonalties would constitute a constant context CS element disconnected from the different-cage context CSs. This constant context could be responsible for at least some pecking CR, and th us for at least some of the sensi tiza tion increment shown by the different-cage and the cage- habituation groups.
That the administration of Apo does actually lead to a detectable interoceptive state has been demon- strated recently in an as yet unpublished drug discrimination experiment of ours. In this experiment the same hungry pigeons learned to peck one key for food reward after having been injected with saline, and to peck another, alternative key after having been injected with Apo (cf. Jarbe, 1984; Tang and Code, 1989). It is known that dopamine receptors are present in the retina of birds (cf. Rohrer and Stell, 1995; see also Drumheller el al., 1995: mammals).
When these receptors are activated by a systemic Apo administration they can be expected to lead to the generation of a stimulus state that is likely to modify the perception of exteroceptive visual stimuli (Djam- goz and Wagner, 1992). Apo administration can thus be expected to create an own CS to which an own CR can be attached (we referred to this process above) but, furthermore, to tinge the perception of environ- mental context CSs, so that they are no longer effectively equivalent with the same exteroceptive context CSs perceived in the absence of the drug. This latter process explains, as already expounded in the Introduction, why sensitized pigeons, when treated with saline to test for the presence of a pecking CR Apo, exhibit a response that is only a small fraction of the sensitization increment, as in fact replicated in this and the persistence experiment. The then effective environmental context CSSalillc is necessarily only very partially equivalent to the total environ- ment-drug CSApo that acted during the sensitization training, and can thus only command a CRSalillc fraction out of the total CRApo that is reflected by the sensitization increment (Godoy and Delius, 1999).
Sensitization persistence
Although not particularly supporting the condition- ing account, the longer-term persistence of the sensitization to Apo found in this experiment is, at the same time, not at all contrary to it. The slow decay of sensitization, to about half in 2 years, can be compared with a similarly slow rate of forgetting, found when pigeons were conditioned to visual
stimuli (Vaughan and Greene, 1984; Fersen and Deli us, 1989). This is a durability that goes well beyond the persistence of the few weeks so far documented in rodents (Mattingly et aI., 1989;
Battisti et al., 1999). Beyond this, we note that, as already remarked above, the pecking response of sensitized pigeons exposed to the environmental context after saline administration was again con- siderably weaker than, but nevertheless proportional to, the response shown when exposed to the same context after Apo administration.
Partially similar conditioning accounts as those considered here have been considered by several authors in connection with the sensitization to different psychostill1ulant drugs (including Apo) with respect to various responses in different species and strains of rodents. These accounts have been suitably reviewed, usefully unified and contrasted with alter- native accounts by Anagnostaras and Robinson (1996; see below for some more recent studies).
However, it is also true that they could not accommodate some of the results they reviewed with the classical conditioning account they distilled. But then, that account did not emphasize the role of two factors that we consider to be crucial: the stimulus generalization that can occur between contexts and the interoceptive stimulus effects that drugs can produce. Concerning the latter effects, Stephens et al. (2000) have recently shown in connection with amphetamine sensitization in rats that an interocep- tively acting drug (chlordiazepoxide), which initially does not yield an overt behavioural response, can come to act as a potent context CS controlling a sizeable CR (see also Pert ef aI., 1990; Kim et al., 1999). Similarly, essential US-derived contributions to finally effective CS have been reported to occur in more conventional varieties of classical conditioning in rats (Bouton, 1993). Within another framework, but also in rats, Dworkin and Dworkin (1995) have documented that internal physiological events of one and the same quality can act both as a CS (small rise in blood pressure) and as a US (large rise in blood pressure). When repeatedly occurring in sequence, they yield a kind of autoconditioning, in the sense that compensatory falls in blood pressure, at first only occurring as URs in response to large rises in blood pressure, come to be triggered as a CR by small rises in blood pressure.
We must consider briefly the extent to which our expanded conditioning squares up with the sensitiza- tion results obtained with Apo in rodents. Carey (1986) found that the contraversive and ipsiversive rotation induced by Apo and amphetamine in unilaterally 6-hydroxydopamine substantia nigra lesioned rats could be dir-rerentially conditioned to
two different environments, in the sense that the rats showed congruent conditioned rotational responses when placed untreated in the two different condi- tioned stimulus contexts. This result fits in easily with the conditioning hypothesis. Moller et al. (1987a, b) describe that after sensitization (conditioning) with Apo in a salient context, rats exhibited more con- ditioned sniffing, licking and gnawing (high-dose- treated animals) or resting and napping (Iow-dose- treated animals) when tested in that context after saline administration and after Apo administration than after a control (pseudo-conditioning) treatment.
As the authors themselves note, these results are consonant with a conditioning account. Tirelli and Heidbreder (I 999a, b) found that sensitization to Apo with reference to a climbing response in mice was mainly context dependent. The authors themselves ascribe the context-dependent sensitization to a Pavlovian conditioning process and conclude that it is definitely not due to a familiarization with the training context. Battisti et al. (J 999, 2000) described that, after a single high dose of Apo (or ampheta- mine), mice became sensitized with respect to a stereotypical repetitive head and forelimb response to a later lower-dose challenge in the same, but not in a different, environmental context. But they also found that after three daily repeated high doses of Apo (or amphetamine) given in the same environmental context, sensitization was later shown in response to the lower dose of Apo (or amphetamine) in both the same and a different context. The context-dependent form of sensitization was found to be susceptible to extinction, but the context-independent extinction was found to be resistant to extinction. Clearly, context-dependent sensitization agrees with a con- ditioning account. The development of an apparently context-independent sensitization with repeated drug administrations could be due to the interoceptive context induced by the Apo itself having acquired most of the control over the response. Mattingly and Gotsick (1989) found a partial context dependence for sensitization with reference to locomotor activity triggered by Apo in rats, but could not observe a locomotor CR when the rats were exposed to the relevant environment after being injected with saline.
This might also indicate a strong conditional contribution of the interoceptive drug context. In contrast, Mattingly ef al. (1997) found that rals sensitized to Apo with respect to a locomotor response in an activity drum or on a running wheel and later tested in a wheel and a drum did not reveal any context dependency. This argues against a conditioning account unless some extreme stimulus generalization was at play. However, only rats Apo- sensitized in a mobile wheel, bUl not rats Apo-
sensitized in a locked wheel, exhibited a strong locomotor response when tested in a mobile wheel.
Although the authors explain both of these results with a non-con text-dependent response enhancement due to an instrumental conditioning process (cf.
Willner et aI., 1992), the latter result can also be interpreted as indicating a context dependency, inasmuch as a moving and a locked wheel undoubt- edly constitute two quite different kinds of sensory contexts.
A more cursory reading of the many recent studies on the sensitization to amphetamine and cocaine, both indirect and unspecific agonists of dopamine, indicates that many, but by no means all, report the occurrence of environmental context dependent sensitization attributed to classical conditioning (e.g.
Carey and Gui, 1997; Lienau and Kuschinsky, 1997;
Badiani et at., 1998; Browman et al., 1998; Nakamura et aI., 1999; Adams et al., 2000; Arvanitogiannis et al., 2000; Crombag et aI., 2000, 2001). Instances of environmental context independent sensitizations, of environmental context novelty dependent sensitiza- tions, of relatively weak saline-elicited CRs and even of sensitization dissociated from corresponding CRs are also reported. Some, but possibly not all, of these findings might be explained by appealing to the effect of varying interoceptive contexts elicited by the drugs, and by a consequent enhancement or suppression of generalization processes. The difficulty is that the studies that have yielded results that are not easily accounted for by conditioning have often employed paradigms that are not particularly suitable for assessment of that circumstance. A cautious, but perhaps realistic, conclusion might be to assume that the sensitizations to diverse drugs under diverse regimes in diverse animals and gauged by diverse behaviours are being brought about by several different mechanisms, with classical conditioning being one of them. However that may be, the present results continue to support the hypothesis that in pigeons and with respect to the pecking reaction, sensitization to Apo is due princi- pally to a combined Pavlovian conditioning to the external context provided by the surrounding envir- onment and to an inner context supplied by the drug i tsel f.
Acknowledgements
This research was supported by research grants from the Deutsche Forscllllngsgemeinschaft, Bonn. We thank Ines Krug and Anke McLintock, Konstanz, for varied assis- tance, Dr Adriana Godoy, Midaga, for much advice, Jennifer Lee, KOl1stanz, for improvements to language and Dr Julia Delius, Berlin, for bibliographic and editorial help.
